LOCUS Exported 4546 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Zeocin selection) with mEos4b. ACCESSION . VERSION . KEYWORDS pMaCTag-Z17 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4546) AUTHORS Fueller J, Herbst K, Meurer M, Gubicza K, Kurtulmus B, Knopf JD, Kirrmaier D, Buchmuller BC, Pereira G, Lemberg MK, Knop M TITLE CRISPR-Cas12a-assisted PCR tagging of mammalian genes. JOURNAL J Cell Biol. 2020 Jun 1;219(6). pii: 151766. doi: 10.1083/jcb.201910210. PUBMED 32406907 REFERENCE 2 (bases 1 to 4546) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4546 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..744 /codon_start=1 /product="fixation-resistant truly monomeric green-to-red photoswitchable fluorescent protein (Paez-Segala et al., 2015)" /label=mEos4b /note="derived from mEos2" /translation="VSAIKPDMRIKLRMEGNVNGHHFVIDGDGTGKPYEGKQTMDLEVK EGGPLPFAFDILTTAFHYGNRVFVKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNAR NDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLL EGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHAIEILSHDKDYNKVKLYEHAVAHSGLPD NARR" polyA_signal 751..872 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(788..807) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 842..861 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 879..912 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 919..1235 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1086..1221 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1148..1167 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" regulatory 1242..1251 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1248..1622 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 1632..1856 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1863..2103 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1863..1883 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2034..2053 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2113..2146 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2183..2202) /label=T7 /note="T7 promoter, forward primer" promoter complement(2184..2202) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2289..2306) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2460..3048) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2540..2559) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3219..4079) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 3842..3861 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4080..4184) /gene="bla" /label=AmpR promoter primer_bind 4252..4270 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4308..4330) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4430..4449 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 4530..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 4530..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tccgtgagtg cgattaagcc agacatgagg atcaaactcc gtatggaagg caacgtaaac 121 gggcaccact ttgtgatcga cggagatggt acaggcaagc cttatgaggg aaaacagacc 181 atggatcttg aagtcaaaga gggcggacct ctgccttttg cctttgatat cctgaccact 241 gcattccatt acggcaacag ggtattcgtg aaatatccag acaacataca agactatttt 301 aagcagtcgt ttcctaaggg gtattcgtgg gaacgaagct tgactttcga agacgggggc 361 atttgcaatg ccagaaacga cataacaatg gaaggggaca ctttctataa taaagttcga 421 ttttatggta ccaactttcc cgccaatggt ccagttatgc agaagaagac gctgaaatgg 481 gagccctcca ctgagaaaat gtatgtgcgt gatggagtgc tgacgggtga tattgagatg 541 gctttgttgc ttgaaggaaa tgcccattac cgatgtgact tcagaactac ttacaaagct 601 aaggagaagg gtgtcaagtt accaggcgcc cactttgtgg accacgccat tgagatttta 661 agccatgaca aagattacaa caaggttaag ctgtatgagc atgctgttgc tcattctgga 721 ttgcctgaca atgccagacg atgaactagt aacttgttta ttgcagctta taatggttac 781 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 841 tgtggtttgt ccaaactcat caatgtatct tagtcgatat aacttcgtat agcatacatt 901 atacgaagtt atgtcgacgg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 961 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag 1021 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 1081 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 1141 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt 1201 gaggaggctt ttttggaggc ctaggctttt gcaaagaatt cgccaccatg gccaagttga 1261 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg 1321 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg 1381 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct 1441 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga 1501 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg 1561 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact 1621 gactgcagcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 1681 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 1741 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 1801 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggctcg 1861 aggagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag 1921 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta 1981 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc 2041 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag 2101 gacgaaacac cgataacttc gtatagcata cattatacga agttatggta ccgatgcagc 2161 tagcccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt 2221 aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 2281 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 2341 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 2401 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 2461 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 2521 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 2581 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 2641 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 2701 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 2761 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 2821 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 2881 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 2941 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 3001 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 3061 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 3121 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 3181 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 3241 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 3301 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 3361 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 3421 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 3481 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 3541 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 3601 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 3661 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 3721 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 3781 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 3841 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 3901 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 3961 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 4021 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 4081 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 4141 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 4201 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 4261 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg 4321 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt 4381 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag 4441 cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa 4501 tacataacct tatgtatcat acacatacga tttaggtgac actata //